Collagen fibrils, proteoglycan aggregates and glycoproteins are critical components of the cartilage extracellular matrix that, collectively, resist compression and the tensile and shear forces that are generated during articulation. Heineg.ang.rd and Oldberg, FASEB J. 3:2042-2051 (1989); Mayne and Brewton, Cartilage Degradation: Basic and Clinical Aspects (Woessner, J. F. and Howell, D. S., eds.) Marcel Dekker, Inc., New York, pp. 81-108 (1993). Mutations in cartilage matrix genes that affect the biosynthesis, assembly or interactions between these various matrix components may contribute to degradation of the cartilage matrix and the loss of normal cartilage function. Mutations in human collagens have been shown to cause a series of chondrodysplasias ranging in severity from lethal achondrogenesis type II to Stickler arthro-ophthalmopathy and early onset familial osteoarthritis (reviewed by Spranger et al., Eur. J. Pediatr. 153:56-65 (1994); Vikkula et al., Ann. Medicine 26:107-114 (1994); Prockop and Kivirikko, Annu. Rev. Biochem. 64:403-434 (1995)).
Analyses of type IX collagen demonstrate this molecule is located on the surface of type II collagen-containing fibrils in hyaline cartilage and other tissues, including the vitreous humor (reviewed by Brewton and Mayne, Extracellular Matrix Assembly and Structure (Yurchenco, P. D., Birk, D. E., Mecham. R. P., eds) Academic Press, Inc., San Diego, pp. 129-170 (1994)). Type IX collagen is a heterotrimer composed of three polypeptide subunits: .alpha.1(IX), .alpha.2(IX) and .alpha.3(IX), that are products of distinct genes and that contain alternating non-triple-helical or noncollagenous domains (NC1-4) and triple-helical or collagenous domains (COL1-3). The three polypeptide subunits are assembled into a mature collagen molecule with the structure .alpha.1(IX).alpha.2(IX).alpha.3(IX) (van der Rest and Mayne, Structure and Function of Collagen Types (Mayne, R. and Burgeson, R., eds.) Academic Press, Orlando, Fla., pp. 195-221 (1987). In addition to type II and type IX collagen, hyaline cartilage from a variety of sources also contains significant amounts of at least three other collagen molecules, types VI, X and XI. Thomas et al., Ann. Rheumat. Diseases 53:488-496 (1994); Mayne and Brewton, Cartilage Degradation: Basic and Clinical Aspects (Woessner, J. F. and Howell, D. S., eds) Marcel Dekker, Inc., New York, pp. 81-108 (1993). Type XI collagen, like type IX collagen, is a heterotrimer composed of three different polypeptide subunits, .alpha.1(XI), .alpha.2(XI), and .alpha.3(XI). Collagen types XII and XIV were also isolated from bovine articular cartilage. Watt et al., J. Biol. Chem. 267:20093-20099 (1992).
Native type IX collagen molecules interact with type II collagen molecules in a highly specific manner so that the domains NC1, COL1, NC2, COL2 and NC3 lie along the surface of the collagen fibril. The interactions between type IX and type II collagen are stabilized by multiple covalent crosslinks derived from specific lysine residues. See van der Rest and Mayne, J. Biol. Chem. 263:1615-1618 (1988); Shimokomaki et al., Ann. N.Y. Acad. Sci. 580:1-7 (1990); Wu et al., J. Biol. Chem. 267:23007-23014 (1992). The periodic localization of type IX collagen along type II collagen fibrils can be readily visualized by rotary shadowing because the collagenous domain COL3 and the large globular domain NC4 project from the surface of the fibril. Vaughan et al., J. Cell Biol. 106:991-997 (1988); Shimokomaki et al., Ann. N.Y. Acad. Sci. 580:1-7 (1990).
The genes encoding the three chains of type
In one embodiment IX collagen are excellent candidates for chondrodysplasias and degenerative disorders that affect the joints and/or vitreous humor because type IX collagen is a significant structural molecule in both of these tissues. Therefore, cloning of the genes encoding the three type IX collagen subunits has been the object of intensive research. Muragaki et al., Eur. J. Biochem. 192:703-8 (1990), presented the complete cDNA sequence of both alternative transcripts from the human .alpha.1(IX) gene. The majority of the human .alpha.2(IX) collagen cDNA sequence was reported by Perala et al., FEBS Lett. 319:177-80 (1993), and the sequence was completed by Warman et al., Genomics 23:158-62 (1994). The complete human sequence for the .alpha.3(IX) subunit has been unavailable.
Experiments utilizing transgenic mice suggest that type IX collagen plays an important role in maintaining the integrity of hyaline cartilage. Animals that either express a minigene carrying a deletion in the .alpha.1(IX) chain (Nakata et al., Proc. Natl. Acad. Sci. U.S.A. 90:2870-2874 (1993)) or that carry disrupted .alpha.1(IX) genes (Fassler et al., Proc. Natl. Acad. Sci. U.S.A. 91:5070-5074 (1994)) develop degenerative joint disease that resembles human osteoarthritis. The importance of type IX collagen in human disease was verified by the identification of a mutation in COL9A2 (Muragaki et al., submitted for publication, (1995)) that results in the skipping of exon 3 and that causes Multiple Epiphyseal Dysplasia (EDM2).
Although a full length cDNA clone encoding the .alpha.3 subunit of chicken type IX collagen was reported several years ago (Brewton et al., Eur. J. Biochem. 205:443-449 (1992)), attempts to obtain the complete structure of the human .alpha.3(IX) collagen subunit gene sequence were unsuccessful. In fact, several publications mention the unavailability and need for the human .alpha.3(IX) collagen gene sequence. See Perala et al., FEBS Lett., 319:177-180 (1993); Perala et al., J. Biol. Chem., 269:5064-71 (1994); Warman et al., Genomics 23:158-62 (1994). The coding sequence and chromosomal location of the .alpha.3(IX) gene was necessary in order to determine if collagen related diseases in humans were caused by alterations in .alpha.3(IX) collagen protein subunit sequence or its production. Moreover, it is impossible to express recombinant human .alpha.3(IX) collagen and trimeric type IX collagen protein for therapeutic applications without the primary amino acid sequence of this collagen.